Process for the preparation of L-amino acids from their racemic N-acetyl-D,L derivatives by enzymatic resolution by means of isolated, recombinant enzymes.
The production of the nonproteinogenic amino acid L-PPT (L-phosphinothricin) by resolution with high purity and yield has previously only been described by cleavage of phenacetylphosphinothricin with penicillin G acylase from Escherichia coli (DE-A-3048612). The synthesis of phenacetyl-PPT, however, is more complicated and more expensive in comparison with N-Ac-PPT. However, penicillin G acylase has no specificity for aliphatic acyl radicals and thus also no specificity for N-Ac-PPT. Other known acylases likewise have no or only a low substrate specificity for N-Ac-PPT and have previously only been employed in microbial biotransformations without purification of the enzymes (as described, for example, in DE-A-2939269). On account of this, only very low space/time yields were achievable. The patent application EP-A-0382113 discloses the L-specific cleavage of N-Ac-PPT carboxylic acid esters by acylase 1. Even this enzyme, however, has no specificity for the free carboxylic acid and therefore necessitates esterification as an additional synthesis step in the preparation of the substrate.
The patent application DE-A-1 9652284 describes the specific isolation of microbial deacetylases from soil samples with specificity for N-acetylamino acids, preferably N-acetylphosphinothricin (N-Ac-PPT), and the cloning of the corresponding genes from Stenotrophomonas sp. and Comamonas acidovorans. 
On account of the sequence homologies found and the substrate specificity tests carried out, it was possible to show that the deacetylases described belong to the group of hippurate hydrolases (EC 3.5.1.32), whose natural substrate is N-benzoylglycine, an amino acid derivative having an aromatic N-acyl function. It was therefore interesting that these enzymes can also accept N-acetylated amino acids, in particular N-acetylphosphinothricin, as a substrate, these being amino acid derivatives having aliphatic N-acyl functions. The patent applications DE-A-2939269 and DE-A-2717440 disclose that the herbicidal action of racemic phosphinothricin emanates from its L enantiomer (L-PPT) alone.
In this connection, it was interesting that the deacetylases found exclusively cleave the L-enantiomers of N-acetyl-PPT, and of N-acetyl derivatives of some proteinogenic amino acids, with high specificity. These enzymes are therefore excellently suited to the preparation of L-amino acids, in particular of the herbicidal active compound L-phosphinothricin, from their racemic N-acetyl derivatives according to the principle of resolution, as is described, for example, in the patent applications EP-A-0304021, DE-A-2939269 and DE-A-3048612. Significant disadvantages of the processes in the three patent applications described above, however, consist in the fact that (1) only low substrate concentrations (about 0.5% in the case of the patent application DE-A-2939269) can be employed, on account of which industrial suitability is to be assessed as low, that (2) reaction is carried out with nonisolated enzymes, on account of which the problems of side reactions and subsequent purification steps cannot be solved or can only be solved very cost-intensively and that (3) cost-intensive product preparation is necessary (as in the case of the patent application DE-A-3048612).
The object on which the invention is based lies in expressing one or more novel deacetylases (such as already characterized in DE-A-19652284) in a suitable form and amount, and in making it possible with the aid of these enzymes to prepare L-PPT and some proteinogenic L-amino acids from the chemically very easily accessible racemic N-acetyl-D,L derivatives by resolution with high yield and enantiomeric purity.
In the case in which the enzymatic activity is already approximately known, and in which cloning of the nucleic acid sequence encoding the enzyme has also already been carried out, the first operation consists in transferring the appropriate nucleic acid fragment to a suitable vector in order then either to carry out overproduction in a suitable bacterial strain, and/or in order to isolate the protein after the overexpression. The enzyme isolated in this way can either be used directly for the enzymatic reaction or else attached to a matrix by means of suitable coupling groups.
A first enzymatic test is advisable in order to obtain general confirmation as to whether the desired reaction is proceeding. All further steps serve to optimize the parameters with respect to the reaction specificity (based on starting material and product), the reaction rate, the reaction efficiency, the half-life of the enzyme employed, and the possible substrate concentrations. In the case that individual parameters do not correspond exactly to the requirements, one is able to carry out suitable changes to the nucleic acid sequence encoding the enzyme, which results in a further optimization of the natural enzyme present.
The cloning of individual deacetylases used has already been described in the application DE-A-1 9652284. The nucleic acid fragments coding for the appropriate deacetylases were recloned in the appropriate expression vectors as carried out further below in Example 1 in order thus to ensure a necessary extent of unequivocal substrate specificity and the absence of side reactions. Thus, although it was possible in the application DE-A-1 9652284 to show that N-acetyl-L-PPT is deacetylated, it was not possible to show that N-acetyl-D-PPT is not also deacetylated. The exclusive reaction of N-acetyl-L-amino acids and N-acetyl-L-PPT, however, is an elementary precondition for the industrial use intended here.
The invention relates to a process for the preparation of proteinogenic or nonproteinogenic L-amino acids from their racemic N-acetyl-D,L derivatives, which comprises
(a) selectively deacetylating N-acetyl-L derivatives of the corresponding L-amino acids by an enzymatic resolution by means of isolated, recombinant enzymes, while N-acetyl-D derivatives of the corresponding D-amino acids are not deacetylated and
(b) separating the deacetylated L-amino acids obtained preparatively from the nondeacetylated N-acetyl-D derivatives and/or the incompletely deacetylated N-acetyl-L derivatives.
The invention relates in particular to a process for the preparation of L-phosphinothricin (L-PPT) from N-acetyl-D,L-phosphinothricin by enzymatic resolution by means of isolated, recombinant enzymes.
The invention furthermore relates to a process for the preparation of L-glutamic acid, L-histidine, L-leucine, L-glutamine and/or L-phenylalanine from their corresponding N-acetyl-D,L derivatives by enzymatic resolution.
The invention furthermore relates to a process which comprises carrying out the enantioselective production of one or more L-amino acids from their corresponding racemic N-acetyl-D,L derivatives using one or more deacetylases from the group consisting of the hippurate hydrolases, and in particular carrying out the enantioselective production of L-phosphinothricin, L-glutamic acid, L-histidine, L-leucine, L-glutamine and/or L-phenylalanine from their corresponding racemic N-acetyl-D,L derivatives using one or more deacetylases from the group consisting of the hippurate hydrolases.
In particular, the invention relates to a process which comprises carrying out the enantioselective production of one or more L-amino acids from their corresponding racemic N-acetyl-D,L derivatives using the enzymes deac1 from Stenotrophomonas sp. and/or deac2 from Comamonas acidovorans (both deacetylases are described in DE-A-19652284), and in this context very particularly the processes which comprise carrying out the enantioselective production of L-phosphinothricin, L-glutamic acid, L-histidine, L-leucine, L-glutamine and/or L-phenylalanine from their corresponding racemic N-acetyl-D,L derivatives using the enzymes deac1 from Stenotrophomonas sp. or/and deac2 from Comamonas acidovorans (both deacetylases are described in DE-A-19652284). Both enzymes have a significant sequence homology, which results in an identical or at least similar function in the cleavage of N-acetyl-D,L derivatives.
In addition, the invention relates to the use of the recombinant deacetylases produced as biocatalysts which allow the appropriate reactions to be carried out using high substrate concentrations and/or with the attainment of high space/time yields, in particular using the recombinant deacetylases in immobilized form.
The invention furthermore relates to the carrying-out of the processes described above at reaction temperatures of approximately 25xc2x0 C. to 65xc2x0 C., preferably at reaction temperatures of approximately 30xc2x0 C. to 45xc2x0 C. and particularly preferably at reaction temperatures of approximately 35xc2x0 C. to 40xc2x0 C.
In addition, the invention relates to the carrying-out of the processes described above at substrate concentrations of approximately 10 mM to 1500 mM, preferably at substrate concentrations of greater than 50 mM, particularly preferably at substrate concentrations of greater than 250 mM and very particularly preferably at substrate concentrations of greater than 500 mM.
The invention furthermore relates to the separation of the L-amino acid or of the L-PTC produced with the aid of the steps described above from the corresponding N-acetyl-D,L derivatives, which have either not been reactedxe2x80x94as in the case of N-acetyl-D derivativexe2x80x94or not been completely reactedxe2x80x94as in the case of N-acetyl-L derivative.
In this context, utilizable processes are the use of ion-exchange chromatography on an acidic ion exchanger or else the extraction of the N-acetyl-D,L derivatives by means of an organic solvent, such as, for example, of methyl isobutyl ketone, the L-amino acid or the L-PTC produced above passing into the aqueous phase, from which it is then concentrated by drying.